The long-range goal of this research is to dissect the contributions of uterine macrophages to the complex processes required for successful reproduction. Substantial progress was made during the initial funding period in understanding the distribution, chemoattraction, differentiation, activation and cytokine production of these powerful, multifunctional cells. The research proposed in this application is directed toward defining the unique conditions in healthy and diseased uteri that govern functional heterogeneity of resident and chemoattracted macrophages. Data collected to date strongly support the postulate that female sex steroid hormones, estrogens and progesterone, bear the overall responsibility for activating uterine macrophages and regulating their production of three potent effector molecules, tumor necrosis factor-alpha (TNF), nitric oxide (NO) and transforming growth factor-beta-1 (TGF-beta-1). Whether hormones accomplish this by direct binding to effector molecule genes or indirectly by stimulating macrophage-targeting growth factors in nearby cells is unknown. Nor has it been determined how hormonally-conditioned macrophages might respond to lipopolysaccharide (LPS) during gram negative bacterial infections. Because each of these effector molecules has the power to alter the course of pregnancy, three Specific Aims have been designed to dissect mechanisms regulating their production in mouse uterine macrophages. The goal of Specific Aim 1 is to define conditions of uterine macrophage activation by determining (a) whether female sex steroid hormones or hormonally-stimulated growth factors regulate macrophage interferon-gamma receptor (IFN-gamma-R) expression, (b) which if any cells in cycling and pregnant mouse uteri express the IFN-gamma gene, and (c) whether hormone conditioning influences IFN-gamma-mediated macrophage activation. In Specific Aim 2, activation for effector molecule production by female sex steroid hormones is dissected from activation by hormonally-stimulated growth factors. The goal of Specific Aim 3 is to determine whether special conditions of pregnancy result in altered sensitivity of macrophages to activation by LPS. Methods used to analyze gene regulation will include northern blot and in situ hybridization experiments (IFN-gamma, jun family, TNF, iNOS, TGF-beta-1 mRNA), electrophoretic mobility-shift assays (NF-kappa-B, jun family proteins), enzyme immunoassays, immunocytochemistry and western blotting as well as bioassays to analyze TNF, iNOS/NO and TGF-beta-1 proteins. Three model systems, 1) uterine macrophages in situ, 2) macrophages selectively harvested from uteri, and 3) macrophage cell lines, will be used for in vivo and in vitro experiments. The results of this research are expected to provide new insights into the regulatory mechanisms operating in the uterus that govern the ability of macrophages to modulate myometrial relaxation, restrict trophoblast invasion, protect against growth of malignant cells, and contribute to infection-associated preterm labor.